The recent upsurge in the degree of drug abuse has led to the necessity for rapid, reliable and efficient methods for detecting the presence of drugs, their metabolites or derivatives in human body fluids such as plasma, urine, etc. In recent years, numerous methods have been developed for the detection of a variety of drugs subject to abuse, such as morphine, barbiturates, amphetamines, lysergide, nicotine, diphenylhydantoin, chloradiazepoxide and cannabinoids.
The following patents describe these methods: U.S. Pat. No. 3,766,162, Spector; U.S. Pat. No. 3,775,536, Spector et al; U.S. Pat. No. 3,799,741, Williams; U.S. Pat. No. 3,882,245, Spector et al; U.S. Pat. No. 3,843,696, Wagner et al; U.S. Pat. No. 3,853,987, Dreyer; U.S. Pat. No. 3,867,366, Rubenstein et al; U.S. Pat. No. 3,878,187, Schneider et al; U.S. Pat. No. 3,879,262, Schuurs et al; U.S. Pat. No. 3,884,898, Schneider; U.S. Pat. No. 3,888,864, Cleeland et al; U.S. Pat. No. 3,952,091, Grunberg et al; U.S. Pat. No. 3,966,744, Goldstein et al; U.S. Pat. No. 3,888,866, Lente et al; and, U.S. Pat. No. 3,690,834, Goldstein et al.
One of the most convenient, efficient and accurate methods for the detection of small amounts of organic compounds in fluids is the so-called radioimmunoassay method. See Radioimmunoassay Methods, Churchill, Livingston, London, 1971, and Yalow et al, Journal of Clinical Investigation, Vol. 39, pg. 1157, 1960.
Radioimmunoassay (RIA) is a term employed to describe any of several methods for determining small concentrations of substances in biological fluids based on the utilization of radioactively labeled substances which form immuno-chemical complexes with antibodies to that substance. Various RIA techniques are known for measuring concentrations of both antibodies and substances for which there exist antibodies. The RIA of a substance for which there exists antibodies specific to that substance is based on the observation that a known amount of that substance which has been radioactively labeled will compete equally with an unknown amount of that substance which is unlabeled for a limited number of complexing sites on antibodies specific to the substance.
RIA is especially suited for the detection of extremely minute amounts of substances in biological fluids and would, therefore, be extremely valuable in the detection of drugs or their metabolites in the biological fluids of the body. With conventional analytical methods, it is extremely difficult to measure the minute amounts of such metabolites which occur in human body fluids due to the intake of the drugs.
As noted above, RIA procedures are based upon the characteristics that an antibody binds equally to labeled or unlabeled antigen. The concentration of the non-labeled form in the solution determines the relative amount of labeled or non-labeled antigen which will bind to antibody. By maintaining the concentration of antibody and labeled antigen constant and conducting the RIA procedure using a series of known amounts of non-labeled antigen, a standard curve can be constructed. Subsequently, when an unknown amount of antigen in a serum or other biological sample is reacted in the same way, its concentration can be determined by relating the value obtained to the standard curve.
The technique may be represented by the following scheme: ##EQU1## where, Ag is the antigen to be assayed
Ag* is the labeled antigen and PA1 Ab is the anti body
The amount of Ab is arranged to be insufficient to react with all of Ag+Ag*. The amount of Ag* added and Ag present in the standards or the unknown sample will compete for the limited binding sites on Ab. If Ag* is separated from Ag*Ab in the above reaction and the level of activity of each separated part measured, then their individual values or their ratios would be related to the amount of Ag present in the standards or the sample. By the use of standard preparations of Ag, a calibration curve is generated.
To date, however, no completely satisfactory method has been proposed for the radioimmunoassay of cocaine, its derivatives or metabolites in body fluids.
Recent studies have shown that cocaine is rapidly metabolized in man. The principal metabolite that appears in urine is benzoylecgonine (BE) which is found along with smaller amounts of ecgonine (E). Cocaine itself is found in very low concentrations, if at all. Cocaine and its metabolites have been analyzed by thin-layer (TLC) and gas chromatography (GLC) and more recently by the Enzyme Multiplied Immunoassay Technique (EMIT). U.S. Pat. No. 3,888,866 describes a spin label immunoassay for cocaine derivatives.
The major disadvantages associated with TLC, GLC and EMIT assays are their lack of sensitivity and their lack of susceptibility to automation. Moreover, EMIT assays are subject to interference due to enzyme poisons present in many biological specimens, particularly, endogenous lysozyme in some human urine specimens.
A further problem which confronts the art is the screening of body fluids for the presence of multiple drugs. If a subject is suspected of having ingested a drug and no clue whatsoever exists as to the nature of the drug, it could be highly advantageous to be able to "screen" the subject's body fluids for a plurality of drugs in a single test, thereby eliminating the necessity of having to run a series of tests designed only to test for the presence of a single drug. Obviously, the provision of a method for at least qualitatively analyzing a body fluid for the presence of multiple drugs, thereby eliminating the presence of a large number of other drugs, would be more economical and of great benefit in controlling and monitoring of drug abuse.
Immunoassay methods have been proposed for detecting the presence of multiple drugs in body fluids; however, these methods depend upon the production of an anti-serum for each particular drug in a separate donor animal and combining the anti-sera in vitro to produce the necessary reagent for the immunoassay method. To date, no method has been provided which enables the production of an anti-sera for a variety of drugs and/or their derivatives from a single donor animal thereby alleviating the disadvantageous time requirements required for the prior art method. Moreover, the maintenance of large numbers of animals and the attendant expense would be eliminated if the anti-serum were derived from a single animal.